WO2001032203A1 - Protease inhibitors as modulators of periodontal wound healing - Google Patents

Abstract

The invention relates to methods for regulating periodontal tissue formation, particularly periodontal tissue attachment, utilising plasminogen activator inhibitors or functional derivatives, equivalents, homologues, analogues or mimetics thereof are described. Further, the invention provides methods for the therapeutic and/or prophylactic treatment of conditions necessitating the up-regulation, inducement or other enhancement of periodontal wound healing such as gingivitis, periodontitis or following gum injuries, and compositions suitable for use in said methods.

Description

PROTEASE INHIBITORS AS MODULATORS OF PERIODONTAL WOUND HEALING

The present invention relates generally to a method of modulating tissue formation and agents useful for same. More particularly, the present invention contemplates a method of up-regulating periodontal tissue formation, particularly periodontal tissue attachment, utilising plasminogen activator inhibitors or functional derivatives, equivalents, homologues, analogues or mimetics thereof. The method of the present invention is useful, ter alia, in the therapeutic and/or prophylactic treatment of conditions necessitating the up-regulation, inducement or other enhancement of periodontal wound healing such as gingivitis, periodontitis or following gum injuries.

Bibliographic details of the publications referred to by author in this specification are collected at the end of the description.

BACKGROUND

Periodontal disease is a general term describing inflammatory disorders of the periodontium.

These range from the relatively benign form known as gingivitis, to the more aggressive forms of early onset periodontitis and rapidly progressive periodontitis. About 8% to 10% of the adult human population in Western countries have marked destructive periodontal disease.

All forms of inflammatory periodontal diseases are associated with bacterial deposits on root surfaces. The inflammation causes connective tissue damage. In gingivitis, removal of the causative agents leads to regeneration of the gingival tissue. However, in periodontitis, a combination of host genetic and environmental factors, including an infection-induced host immune response, can lead to loss of connective tissue attachment to the root surface, bone and ligament loss around the tooth, and the formation of periodontal pockets (spaces between the gum and tooth). Even if the causative inflammation is controlled, and bacteria removed, many of the architectural changes in the hard and soft connective tissues in periodontitis are currently irreversible. Known methods of treating periodontal diseases involve scaling and root planing of the root surfaces with the periodontal pockets in order to reduce the inflammation in the soft tissue wall. In addition, antibiotics can be administered to the periodontal pocket. However, scaling and planing may not result in tissue regeneration, and the use of antibiotics is aimed only at the infectious agent (bacteria) and not at the host immune response. This host response involves the release of a variety of destructive proteolytic enzymes (proteases), including plasmin and metalloproteases, which are the principal agents of bone and tissue destruction. Recently developed treatments aim to block the destructive activity of metalloproteases. However, there are no existing treatments which have the property of promoting full periodontal tissue regeneration and attachment.

Healing or periodontal regeneration is unique, and may be distinguished from healing in other tissues, such as skin for example, because it involves two soft connective tissues (gingiva and periodontal ligament) and two hard, mineralised connective tissues (bone and cementum). However, initial events in periodontal wound healing are similar to skin, namely exudation of plasma constituents and the generation of a provisional matrix of which fibrin is the major constituent. The fibrin matrix is then replaced by granulation tissue, which is in turn replaced by a secondary, collagenous matrix (Clark, 1996). The events which follow have no parallel in skin healing and are believed to involve the coordinated synthesis of growth factors and adhesion proteins, which promote, in a temporally and spatially distinct manner, the adhesion of certain cell types. Progenitor cells (which are poorly understood) differentiate into functional connective tissue cells and thence into cementoblasts. With directional and coordinated formation of new periodontal ligament fibres and alveolar bone, the hard and soft components are assembled and attached to complete the repair (Bartold and Narayanan, 1998).

As will be appreciated from the previous paragraph, the mechanisms involved in tissue regeneration are extremely complex in nature, involving the temporal and spatial interaction of many cells, cellular components, and regulatory molecules and enzymes. In the case of periodontal tissue the complexity may be compounded, or at least differ considerably from that seen in other tissues, due to the environment in which the tissue resides; for example, the mouth cavity is constantly moist, the tissue is exposed to a variety of different digestive enzymes secreted with saliva, any exogenous matter, including food, placed in the oral cavity, and various micro-organisms, and in addition, is subject to constant wear and tear, any one of which factors may effect the process of tissue damage and repair. Consequently, the regeneration of periodontal tissue may be further distinguished from regeneration, or healing, in other tissues, as will the treatment of a condition of periodontal tissue as opposed to the treatment of tissues such as skin.

Generally, in tissue remodelling and cell migration of wound healing plasmin-mediated proteolysis is believed to be important at the biochemical level (Romer, 1996). Plasmin is thought to be of principal importance in the remodelling of fibrin rich matrices, since local extracellular proteolysis is required for the initial removal of the fibrin matrix as well as for the remodelling of the granulation tissue (Clark, 1996). Plasminogen activators (PA) are serine proteases that form part of the complex enzyme cascade involved in fibrinolysis. These enzymes convert plasminogen into plasmin, a trypsin-like serine protease, that is not only responsible for the degradation of fibrin, but also contributes directly and indirectly, via conversion of latent collagenase into active collagenase, to the degradation and turnover of the extracellular matrix (Kruithof, 1988). Plasminogen is activated by either urokinase- type plasminogen activator (u-PA) or tissue-type plasminogen activator (t-PA) (Vassalli, 1991). These catalytic reactions generally take place at the plasma membrane (u-PA) or on a fibrin surface (t-PA). These activating enzymes are produced by a wide range of mesenchymal, epithelial and endothelial cells in response to a variety of cytokines and growth factors.

While the cellular, biochemical, molecular mechanisms involved in the healing of wounds residing in other tissues of the body have been extensively studied and elucidated there has surprisingly been little work done on addressing the precise mechanisms involved in periodontal tissue destruction and regeneration. Accordingly, the knowledge of the precise mechamsms involved and the extent of the involvement of the plasminogen activator system in periodontal wound healing is sparse. Components of the plasminogen activator system (PAS) have been shown to contribute to connective tissue degradation and cell migration (Xiao, et al, 1988) in periodontal tissue however, the functioning and role of the plasminogen activators have only been partially elucidated, and there has been no elucidation of the functioning of the plasminogen activator system in terms of the regulatory mechanisms which are active.

Accordingly, there is a need to identify the precise nature and functioning of the cells and molecules which regulate periodontal tissue damage and repair, if the development of methods for regulating periodontal tissue formation and attachment are to be achieved.

SUMMARY OF INVENTION

The inventors have now determined that the plasminogen activator inhibitors (herein referred to as "PAIs"), and in particular PAI-1 and PAI-2, are actively involved in the regulation of the plasminogen activator system in periodontal tissues. Elucidation of the location and nature of their functional activity now facilitates the development of methods for regulating, and in particular up-regulating, periodontal tissue formation by regulating the activity of the plasminogen activator system. The consequences of this up-regulation include the improved reattachment or adhesion of periodontal tissue to bone.

The present invention uses a treatment which blocks the destructive activity of plasmin by the administration of PAIs, particularly PAI-1 and/or PAI-2, molecules normally present in ginigival crevicular fluid. The proposed use of PAIs, is based on their ability to promote adhesion (attachment) of newly formed periodontal tissue to bone, a feature not known to be possessed by inhibitors of metalloproteases, for example. Such a treatment has not been described hitherto, and no existing treatment possesses, to the authors' knowledge, these features.

Accordingly, the present invention provides a method of regulating periodontal tissue formation in a mammal, said method comprising administering to said mammal an effective amount of one or more PAIs or functional derivatives, equivalents, analogues, homologues or mimetics thereof for a time and under conditions sufficient to modulate the functional activity of any one or more components of the periodontal tissue formation pathway.

Reference to "periodontal tissue" should be understood as a reference to any tissue located in the oral cavity which functions either directly or indirectly to anchor the tooth to its socket in the jawbone. This includes, for example, the soft tissue which attaches to the root surface, such as connective tissue, or the epithelial cells, fibroblasts, endothelial cells or other stromal cells which may also act to directly anchor the tooth. "Periodontal tissue" also includes reference to cells such as monocytes and macrophages which contribute functionally, for example by secretion of cytokines, to the formation of tissue which anchors the tooth. Reference to "periodontal tissue" should also be understood as a reference to tissue which may be transiently present during periodontal tissue formation. For example, the granulation tissue which is formed in the early stages of periodontal wound healing, and which replaces the fibrin matrix, but which has some components replaced as the tissue is remodelled during epithelialisation and the final attachment of soft tissue to the root surface.

Reference to "tissue" should be understood in its broadest sense and includes reference to whole tissue, tissue fragments, specific cell populations or single cells. Reference to

"periodontal tissue formation" should therefore be understood to indicate the formation of the tissue hereinbefore defined which directly or indirectly functions to anchor the tooth (bone) to its socket (soft tissue). Accordingly, reference to "periodontal attachment (or adhesion)" or "periodontal tissue attachment (or adhesion)" should be taken to mean the joining of hard and soft periodontal tissue elements (as defined above) to form a substantially stable junction, preferably permanent, which is substantially impermeable to fluid.

Reference to "regulation" should be understood as a reference to both up-regulation and down-regulation. Although the prefened method is to up-regulate periodontal tissue formation, the down-regulation of periodontal tissue formation may also be desired under certain circumstances. For example, where uncontrolled, aberrant or otherwise unwanted tissue formation is occurcing. In this regard, it may be necessary to only transiently up- regulate the formation of periodontal tissue, for example for the promotion of wound healing associated with an acute injury, or it may be necessary to regulate the formation of this tissue on a long term basis, for example where a patient is suffering from a chronic disease condition. Reference to "up-regulation" should be understood to include reference to both newly inducing activity or enhancing or prolonging existing activity. Similarly, reference to "down-regulation" should be understood to include reference to both preventing the onset of activity or to partially or fully reducing existing activity.

Accordingly, the present invention more particularly provides a method of inducing, enhancing or otherwise up-regulating periodontal tissue formation in a mammal, said method comprising administering to said mammal an effective amount of one or more PAIs or functional derivatives, equivalents, analogues, homologues or mimetics thereof for a time and under conditions sufficient to modulate the functional activity of any one or more components of the periodontal tissue formation pathway.

Reference to "PAT should be understood as a reference to any PAI or functional derivative, equivalent, homologue, analogue or mimetic thereof. In this regard, the PAI may be of any suitable form, such as a mature molecule, a precursor form of said mature molecule, mutant, polymorphic variant or a derivative, homologue, equivalent, analogue or mimetic thereof which exhibits at least one of the functional activities of said PAI. In a preferred embodiment, said PAI is PAI-1, a glycoprotein of the serine protease inhibitor type, or PAI- 2, which is also a serine protease inhibitor, and exists in both glycosylated and unglycosylated forms (Andreasen, 1990). It should also be understood that the present invention extends to the administration of a nucleic acid molecule, or analogue thereof, which encodes the subject PAI for the purpose of its in vivo expression.

According to this embodiment there is provided the method of inducing, enhancing or otherwise up-regulating periodontal tissue formation in a mammal, said method comprising administering to said mammal an effective amount of PAI-1 and/or PAI-2 or functional derivative, equivalent, analogue, homologue or mimetic thereof for a time and under conditions sufficient to modulate the functional activity of any one or more components of the periodontal tissue formation pathway. Preferably, said components are plasminogen activator and/or plasmin and said modulation is inhibition.

Derivatives include fragments, parts, portions, mutants, and mimetics from natural, synthetic or recombinant sources including fusion proteins exhibiting any one or more of the functional activities of the subject PAI. To the extent that the subject PAI is a protein, derivatives may be derived from insertion, deletion or substitution of amino acids. Amino acid insertional derivatives include amino and/or carboxylic terminal fusions as well as intrasequence insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at least one residue in the sequence has been removed and a different residue inserted in its place. An example of substitutional amino acid variants are conservative amino acid substitutions. Conservative amino acid substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine. Additions to amino acid sequences including fusions with other peptides, polypeptides or proteins.

Homologues of a PAI contemplated herein include, but are not limited to, proteins derived from different species.

Chemical and functional equivalents of PAI should be understood as molecules exhibiting any one or more of the functional activities of PAI and may be derived from any source such as being chemically synthesized or identified via screening processes such as natural product screening.

The derivatives of PAI include fragments having particular epitopes of parts of the entire PAI protein fused to peptides, polypeptides or other proteinaceous or non-proteinaceous molecules. For example, PAI or derivative thereof may be fused to a molecule to facilitate its entry into a cell.

Reference to "derivatives" should also be understood to include reference to analogues. Analogues of PAI contemplated herein include, but are not limited to, modification to side chains, incorporating of unnatural amino acids and/or their derivatives during peptide, polypeptide or protein synthesis and the use of crosslinkers and other methods which impose conformational constraints on the proteinaceous molecules or their analogues.

Derivatives of nucleic acid sequences may similarly be derived from single or multiple nucleotide substitutions, deletions and/or additions including fusion with other nucleic acid molecules. The derivatives of the nucleic acid molecules of the present invention include oligonucleotides, PCR primers, antisense molecules, molecules suitable for use in cosuppression and fusion of nucleic acid molecules. Derivatives of nucleic acid sequences also include degenerate variants.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH4; amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with pyridoxal-5-phosphate followed by reduction with NaBH

The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

The carboxyl group may be modified by carbodiimide activation via O-acylisourea formation followed by subsequent derivitisation, for example, to a corresponding amide. Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4-chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4- nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides. Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to form a 3-mtrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carboethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during protein synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy- 5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid contemplated herein is shown in Table 1.

TABLE 1

Non-conventional Code Non-conventional Code amino acid amino acid

α-aminobutyric acid Abu L-N-methylalanine Nmala α-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmarg aminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylate L-N-methylaspartic acid Nmasp aminoisobutyric acid Aib L-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgln carboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine Chexa L-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucine Nmile

D-alanine Dal L-N-methylleucine Nmleu

D-arginine Darg L-N-methyllysine Nmlys

D-aspartic acid Dasp L-N-methylmethionine Nmmet

D-cysteine Dcys L-N-methylnorleucine Nmnle

D-glutamine Dgln L-N-methylnorvaline Nmnva

D-glutamic acid Dglu L-N-methylornithine Nmorn

D-histidine Dhis L-N-methylphenylalanine Nmphe

D-isoleucine Dile L-N-methylproline Nmpro

D-leucine Dleu L-N-methylserine Nmser

D-lysine Dlys L-N-methylthreonine Nmthr

D-methionine Dmet L-N-methyltryptophan Nmtrp

D-ornithine Dorn L-N-methyltyrosine Nmtyr

D-phenylalanine Dphe L-N-methylvaline Nmval

D-proline Dpro L-N-methylethylglycine Nmetg

D-serine Dser L-N-methyl-t-butylglycine Nmtbug

D-threonine Dthr L-norleucine Nle

D-tryptophan Dtrp L-norvaline Nva

D-tyrosine Dtyr α-methyl-aminoisobutyrate Maib

D-valine Dval α-methyl-γ-aminobutyrate Mgabu

D-α-methylalanine Dmala α-methylcyclohexylalanine Mchexa

D-α-methylarginine Dmarg α-methylcylcopentylalanine Mcpen

D-α-methylasparagine Dmasn α-methyl-α-napthylalanine Manap

D-α-methylaspartate Dmasp α-methylpemcillamine Mpen

D-α-methylcysteine Dmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine Dmgln N-(2-aminoethyl)glycine Naeg

D-α-methylhistidine Dmhis N-(3-aminopropyl)glycine Norn

D-α-methylisoleucine Dmile N-amino-α-methylbutyrate Nmaabu

D-α-methylleucine Dmleu α-napthylalanine Anap D-α-methyllysine Dmlys N-benzylglycine Nphe

D-α-methylmethionine Dmmet N-(2-carbamylethyl)glycine Ngln

D-α-memylornithine Dmorn N-(carbamylmethyl)glycine Nasn

D-α-methylphenylalanine Dmphe N-(2-carboxyethyl)glycine Nglu

D-α-methylproline Dmpro N-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycine Ncbut

D-α-methylthreonine Dmthr N-cycloheptylglycine Nchep

D-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex

D-α-methyltyrosine Dmty N-cyclodecylglycine Ncdec

D-α-methylvaline Dmval N-cylcododecylglycine Ncdod D-N-methylalanine Dnmala N-cyclooctylglycine Ncoct

D-N-methylarginine Dnmarg N-cyclopropylglycine Ncpro

D-N-methylasparagine Dnmasn N-cycloundecylglycine Ncund

D-N-methylaspartate Dnmasp N-(2,2-diphenylethyl)glycine Nbhm

D-N-methylcysteine Dnmcys N-(3 , 3 -diphenylpropy l)glycine Nbhe D-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine Narg

D-N-methylglutamate Dnmglu N-(l-hydroxyethyl)glycine Nthr

D-N-methylhistidine Dnmhis N-(hydroxyethyl))glycine Nser

D-N-methylisoleucine Dnmile N-(imidazolylethyl))glycine Nhis

D-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine Nhtrp D-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate Nmgabu N-methylcyclohexylalanine Nmchexa D-N-methylmethionine Dnm et

D-N-methylornithine Dnmorn N-methylcyclopentylalanine Nmcpen

N-methylglycine Nala D-N-methylphenylalanine Dnmphe N-methylaminoisobutyrate Nmaib D-N-methylproline Dnmpro N-(l-methylpropyl)glycine Nile D-N-methylserine Dnmser N-(2-methylpropyl)glycine Nleu D-N-methylthreonine Dnmthr D-N-methyltryptophan Dnmtrp N-(l-methylethyl)glycine Nval D-N-methyltyrosine Dnmtyr N-methyla-napthylalanine Nmanap D-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acid Gabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine Tbug N-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine Pen L-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine Marg L-α-methylasparagine Masn L-α-methylaspartate Masp L-α-methyl-t-butylglycine Mtbug L-α-methylcysteine Mcys L-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamate Mglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine Mhphe L-α-methylisoleucine Mile N-(2-methylthioethyl)glycine Nmet L-α-methylleucine Mleu L-α-methyllysine Mlys

L-α-methylmethionine Mmet L-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithine Morn L-α-methylphenylalanine Mphe L-α-methylproline Mpro L-α-methylserine Mser L-α-methylthreonine Mthr L-α-methyltryptophan Mtrp L-α-methyltyrosine Mtyr L-α-methylvaline Mval L-N-methylhomophenylalanine Nmhphe N-(N-(2,2-diphenylethyl) Nnbhm N-(N-(3,3-diphenylpropyl) Nnbhe carbamylmethyl)glycine carbamylmethyl)glycine 1 -carboxy- 1 -(2 ,2-dipheny 1- Nmbc ethylamino)cyclopropane

Crosslinkers can be used, for example, to stabilise 3D conformations, using homo- bifunctional crosslinkers such as the bifunctional imido esters having (CH2)_n spacer groups with n= l to n=6, glutaraldehyde, N-hydroxysuccinimide esters and hetero-bifunctional reagents which usually contain an amino-reactive moiety such as N-hydroxysuccinimide and another group specific-reactive moiety.

The PAI molecule may be derived from natural or recombinant sources including fusion proteins or following, for example, natural product screening. The present invention contemplates chemical analogues of PAI capable of acting as agonists or antagonists of PAI. Chemical agonists may not necessarily be derived from PAI but may share certain conformational similarities. Alternatively, chemical agonists may be specifically designed to mimic certain physiochemical properties of PAI. Antagonists may be any compound capable of blocking, inhibiting or otherwise preventing PAI from carrying out its normal biological functions. Antagonists include monoclonal antibodies specific for PAI, or parts of PAI, and antisense nucleic acids which prevent transcription or translation of PAI genes or mRNA in mammalian cells. Agonists and antagonists of PAI should be understood to include any molecule which synergises with PAI to either up-regulate or down-regulate, respectively, its activity. In this regard, reference herein to administering PAI should also be understood to include reference to administering an agent which modulates the functional activity of endogenously produced PAI or which modulates the expression of endogenously produced PAI.

Reference to the "periodontal tissue forming (or, formation) pathway" should be understood as a reference to the sequence of events which lead to periodontal tissue formation and ultimate attachment of soft tissue to the teeth (root, bone).

Without limiting the present invention in any way, the plasminogen activator system is thought to function by the expression of PAs by different cell types in periodontal wound healing and has concerted roles in the formation of fibrin matrix and tissue remodelling. These include dissolution of the fibrin clot, ECM degradation, growth factor mobilization and activation, cell migration into the wound area, angiogenesis, and reepthelialization (Clark, 1996). PAIs are now thought to function in the epithelialisation and reattachment of periodontal tissue and play a significant role in the formation of new periodontal attachment. Specifically, it is thought that during periodontal wound healing the strong expression of PAI-1 and PAI-2 in provisional matrix leads to the formation of the fibrin clot.

The fibrin clot provides an early, primitive form of ECM, which potentiates the migration of inflammatory cells into periodontal sulcus and the lesion. The high amounts of u-PA secreted by granulocytes and macrophages that infiltrate into periodontal wound area, together with the t-PA derived from blood vessel disruption and plasma extravasation, lead to the ultimate dissolution of the fibrin clot. The localization of u-PA, PAI-1 and PAI-2 in new attached fibroblasts and epithelial cells on the root surface indicated that PAs play a significant role in the formation of new periodontal attachment. Accordingly, reference to modulating the functional activity of a "component" of the periodontal tissue forming pathway should be understood as a reference to either directly or indirectly modulating the functional activity of any one or more molecules or cells which function during periodontal tissue formation and attachment. Preferably, said component is an enzyme and even more preferably plasminogen activator and/or plasmin.

Administration of the PAI, in the form of a pharmaceutical composition, may be performed by any convenient means. PAI of the pharmaceutical composition are contemplated to exhibit therapeutic activity when administered in an amount which depends on the particular case. The variation depends, for example, on the human or animal, the PAI chosen and the route of administration. A broad range of doses may be applicable. Considering a patient, for example, from about 0.1 μg to about 10 mg of PAI may be administered per kilogram of body weight per day. For example from about 0.1 μg-5 mg, 10 μg-5 mg or 100 μg-X mg.

Dosage regimes may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily, weekly, monthly or other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the situation. The PAI may be administered in a convenient manner such as by the oral, intravenous (where water soluble), intranasal, intraperitoneal, intramuscular, subcutaneous, intradermal or suppository routes or implanting (e.g. using slow release molecules). With particular reference to use of PAI, these peptides may be administered in the form of pharmaceutically acceptable nontoxic salts, such as acid addition salts or metal complexes, e.g. with calcium, magnesium, zinc, iron or the like (which are considered as salts for purposes of this application). Illustrative of such acid addition salts are hydrochloride, hydrobromide, sulphate, phosphate, maleate, acetate, citrate, benzoate, succinate, malate, ascorbate, tartrate and the like. If the active ingredient is to be administered in tablet form, the tablet may contain a binder such as tragacanth, corn starch or gelatin; a disintegrating agent, such as alginic acid; and a lubricant, such as magnesium stearate. If the active ingredient is to be administered in a gel form, such as sodium methyl cellulose, it could further contain bioadhesive compounds such as chitosan, xanthan gum, methacrylate, polyethylene oxide, carbopol 974P, Noveon (Polycarbophil), poly grip (carboxymethyl cellulose and polyethylene oxide), or pluronic F127.

A further aspect of the present invention is in relation to mammalian disease conditions. The method of the present invention is particularly useful in relation to the treatment of conditions characterised by the damage, breakdown or other form of degradation of periodontal tissue such as that caused by, for example, gingivitis, periondotitis or gum injury caused by, for example, major or minor surgery or infection or pregnancy-related tissue deterioration. As detailed hereinbefore, although the preferred method of the present invention is to up-regulate periodontal tissue formation, the present invention should nevertheless be understood to encompass the down-regulation of periodontal tissue formation where such tissue formation is aberrant or otherwise unwanted.

Accordingly, another aspect of the present invention relates to a method of treating a mammal said method comprising administering to said mammal an effective amount of one or more PAIs or functional derivatives, equivalents, analogues, homologues or mimetics thereof for a time and under conditions sufficient to modulate any one or more components of the periodontal tissue formation pathway.

Preferably, the present invention relates to a method of treating a mammal said method comprising administering to said mammal an effective amount of PAI or derivative, equivalent, analogue, homologue or mimetic thereof for a time and under conditions sufficient to modulate the functional activity of any one or more aspects of the periodontal tissue formation pathway wherein said modulation results in the induction, enhancement or up-regulation of periodontal tissue formation. Preferably said PAI is PAI-1 and/or PAI-2.

Preferably, said components are plasminogen activator and/or plasmin and said modulation is inhibition.

In another aspect the present invention provides a method for the therapeutic and/or prophylactic treatment of a condition in a mammal, which condition is characterised by damage, breakdown or other form of degradation of periodontal tissue, said method comprising administering to said mammal an effective amount of PAI or functional derivative, equivalent, analogue, homologue or mimetic thereof for a time and under conditions sufficient to modulate the functional activity or any one or more aspects of the periodontal tissue formation pathway wherein said modulation results in the induction, enhancement or up-regulation of periodontal tissue formation.

Preferably, said PAI is PAI-1 and/or PAI-2.

Preferably, said components are plasminogen activator and/or plasmin and said modulation is inhibition.

Even more preferably said condition is gingivitis, periodontitis or gum injury.

In yet another aspect the present invention relates to the use of an agent capable of modulating the functional activity of any one or more components of the periodontal tissue formation pathway wherein said agent is PAI or functional derivative, equivalent, homologue, analogue or mimetic thereof.

Preferably, said modulation results in inducement, enhancement or up-regulation of periodontal tissue formation. In still another aspect, the present invention relates to the use of PAI or functional derivative, homologue, analogue, chemical equivalent or mimetic thereof in the manufacture of a medicament for modulating the formation of periodontal tissue.

Preferably, said PAI is PAI-1 and/or PAI-2.

Reference herein to "treatment" and "prophylaxis" is to be considered in its broadest context. The term "treatment" does not necessarily imply that a mammal is treated until total recovery. Similarly, "prophylaxis" does not necessarily mean that the subject will not eventually contract a disease condition. Accordingly, treatment and prophylaxis include amelioration of the symptoms of a particular condition or preventing or otherwise reducing the risk of developing a particular condition. The term "prophylaxis" may be considered as reducing the severity of onset of a particular condition. "Treatment" may also reduce the severity of an existing condition or the frequency of acute attacks.

In accordance with these methods, the PAI defined in accordance with the present invention may be coadministered with one or more other compounds or molecules. By "coadministered" is meant simultaneous administration in the same formulation or in two different formulations via the same or different routes or sequential administration by the same or different routes. By "sequential" administration is meant a time difference of from seconds, minutes, hours or days between the administration of the two types of molecules, These molecules may be administered in any order.

The term "mammal" should be understood as a reference to a human, primate, livestock animal (eg. sheep, pig, cow, horse, donkey) laboratory test animal (eg. mouse, rat, rabbit, guinea pig) companion animal (eg. dog, cat) or captive wild animal (eg. fox, kangaroo, deer). Preferably, the mammal is a human.

An "effective amount" means an amount necessary to at least partly attain the desired response. In yet another further aspect, the present invention contemplates a pharmaceutical composition comprising PAI or functional derivative, homologue, analogue, chemical equivalent or mimetic thereof together with one or more pharmaceutically acceptable carriers and/or diluents. Preferably, said PAI is PAI-1 and/or PAI-2. The PAI molecules are referred to as the active ingredients. The pharmaceutical composition is preferably designed for topical or injectable administration and even more preferably for topical application.

The pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as licithin, by the maintenance of the required particle size in the case of dispersion and by the use of superfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimersal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the various sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile-filtered solution thereof.

When the active ingredients are suitably protected they may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet. For oral therapeutic administration, the active compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 1 to about 80% of the weight of the unit. The amount of active compound in such therapeutically useful compositions in such that a suitable dosage will be obtained. Preferred compositions or preparations according to the present invention are prepared so that an oral dosage unit form contains between about 0.1 μg and 2000 mg of active compound.

The tablets, troches, pills, capsules and the like may also contain the following: A binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, pills, or capsules may be coated with shellac, sugar or both. A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and formulations. The active compound may preferably be incorporated into a topical gel or cream formulation, which may or may not be sterile, and which may contain support-substances such as derivatives of cellulose (sodium methyl- or hydroxy ethyl-) and/or substances to improve absorption such as propylene glycol and/or substances to improve bioadhesion such as chitosan, xanthan gum, methacrylate, polyethylene oxide, carbopol 974P, Noveon (Polycarbophil), polygrip (carboxymethyl cellulose and polyethylene oxide), or pluronic F127. It will be appreciated by those of skill in the art to which this invention relations that any bioadhesives contemplated to be used in the invention should be tested for their compatibility with the active (PAIs). Such testing may be conducted in a manner described herein after (see "Testing of bioadhesive gel formulations for delivery of active to periodontal pocket").

Pharmaceutically acceptable carriers and/or diluents include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. The specification for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active material and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active material for the treatment of disease in living subjects having a diseased condition in which bodily health is impaired. The principal active ingredient is compounded for convenient and effective administration in effective amounts with a suitable pharmaceutically acceptable carrier in dosage unit form as hereinbefore disclosed. A unit dosage form can, for example, contain the principal active compound in amounts ranging from 0.1 μg to about 2000 mg. Expressed in proportions, the active compound is generally present in from about 0.1 μg to about 2000 mg/ml of carrier. In the case of compositions containing supplementary active ingredients, the dosages are determined by reference to the usual dose and manner of administration of the said ingredients.

The pharmaceutical composition may also comprise genetic molecules such as a vector capable of transfecting target cells where the vector carries a nucleic acid molecule capable of modulating periodontal tissue formation. The vector may, for example, be a viral vector.

Further features of the present invention are more fully described in the following non- limiting Figures and/or Examples.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. (a) H&E staining of day 3 section after wound healing. Section shows inflamed cells localized in the sulcus areas and the fibrin clot formed in the wounding areas, as well as the formation of granulation tissue next to the fibrin, (b) PCNA expression in the granulation tissue. The basal epithelial cells and some of the granulation cells were positively stained for PCNA. (c) u-PA expression in the site of inflamed cells. Fibrin clot and inflamed cells were strongly stained for u-PA. (d) PAI-2 expression in the inflamed cell areas. Inflamed cells were stained very strongly for PAI-2 and fibrin clot stained positively. Abbreviations: E= epithelium, D= dentine, IC= inflamed cells, AB= alveolar bone, GT= granulate tissue.

Figure 2. (a) u-PA expression in the cells close to the fibrin clot. Arrows show the strongly positive staining cells (b) u-PA expression in the cells attaching to the root surface. Arrow shows positive cells, (c) In the newly formed epithelium, PAI-2 stained positively in the attached cells. Arrow shows the positive staining, (d). t-PA expression in the newly formed attachment tissue. No obvious staining could been found. Abbreviations: FC= fibrin clot, D= dentine, JE=junction epithelium.

Figure 3. (a) u-PA staimng could be found in the endothelial cells of newly formed blood vessels. Arrows show the positive staining, (b) In granulation tissue (day 5) some granulocytes show positive staining for u-PA. Arrows show the positive staining.

Figure 4. The changes of t-PA concentration in periodontal wound healing fluid. t-PA was increased and reached its peak in the first week, then decreased.

Figure 5. The changes of PAI-2 concentration in periodontal wound healing fluid.

Figure 6. Bioadhesives for Periodontal Delivery of PAI-2. The bioadhesives, their concentration, presence of preservatives, and sterility (bioburden measurement) are listed. The figure summarises the stability of PAI-2 (+) in each bioadhesive. Instability is indicated by (-). Stability in pluronic F127 is temperature dependent. Abbreviations: CMC=carboxymethyl cellulose, HEC=hydroxy ethyl cellulose, PEO= polyethylene oxide, PVP=polyvinyl pyrrolidone, PC=polycarbophil, TPC=total plate count.

Figure 7. PAI-2 Stability in Study Periogels. The activity of PAI-2 in each bioadhesive is measured over time, and is expressed as International Units (IU) per ml, using Spectrozyme assay.

Figure 8. PAI-2 Stability in 17% Pluronic F127. The activity of PAI-2 in pluronic F127 at different temperatures is expressed as in Figure 7. 1 and 2 represent duplicate experiments conducted on different days.

Figure 9. In vitro Release of PAI-2 from Preserved Periogels. PAI-2 in each periogel was placed in the top chamber of a two-chamber vessel, with buffer in the lower chamber. The figure shows the appearance with time of PAI-2 activity in the lower chamber, as a measure of the release of PAI-2 from the gel.

Figure 10. In vitro Release of PAI-2 from Pluronic F127. Conditions as for figure 9, with PAI-2 in different concentrations of Pluronic F127.

DETAILED DESCRIPTION

The invention will become further apparent from the following description which provides details of experiments conducted, and results obtained therefrom, which have led to the present invention. Further, specific examples of formulations according to the invention and methods of administration to a subject in need thereof are provided.

Experimental Protocols Animal model The experimental protocol for this animal study has been approved by the University Animal Experimentation Ethics Committee, University of Queensland. Forty male Lewis rats (12 weeks old and body weight from 230g to 260g on the day of surgery) were used for this experiment. All these rats were caged in pairs and fed with water and nutritious pelleted rat cubes. All surgical procedures were carried out under Nembutal general anaesthesia (Pentobarbitone Sodium, Boehringer Ingelheim Pty Limited, Animal Health Division, 50 Broughton Road, Artarmon NSW 2064, Australia).

To create the surgical wound a full thickness mucoperiosteal flap was raised involving the buccal alveolar bone and the creation of the defect of an alveolar bone crest of the mandibular first molar tooth. The roots were then planed, and all soft tissues were removed from the surface of exposed roots. Using a water-cooled dental bur at a low speed, 2.5 to 3 mm of alveolar bone crest was removed. The surgical areas were washed with 0.9% saline for surgical use. The flaps are then positioned and pressured to the root and left unsutured. Five rats were sacrificed at each group of different wound healing period at day 1, 3, 5, 7, 14, 21, and 28 after the surgery. In addition, five rats served as controls in which no surgery were processed. Before the tissue biopsies were taken the animals were perfused with 5 ml PBS and 5 ml 4% paraformaldehyde.

Tissue preparation

After sacrifice, the mandibles were removed immediately and separated between the middle incisors. The posterior portion of the mandibles, from the first molar to the third molar, were dissected and washed with PBS for 5 minutes, then fixed at 4% paraformaldehyde in PBS at pH 7.4 for 12 hours at room temperature. Tissues were decalcified in 10% EDTA with two changes per week for three to four weeks depending on the degree of decalcification which was checked by X-ray examination. The tissues were then trimmed and dehydrated in graded ethanol from 70%, 90% to 100%, then cleared in toluene and embedded in paraffin. Serial 5 μm sections in a lingual-buccal frontal direction were cut and mounted to glass slides for immunohistochemistry and routine haematoxylin-eosin staining.

Antibodies used for immunohistology

The following monoclonal antibodies were used for immunohistology. Monoclonal mouse anti-tPA IgG (no. 104201; Biopool, Umea, Sweden), which binds to the A-chain in human tissue plasminogen activator is an antibody which reacts with human single-chain tissue plasminogen activator and the proteolytically modified two-chain tissue plasminogen activator. Monoclonal mouse anti-uPA IgG (no. 3689; American Diagnostic) which is directed against a B-chain epitope of human urokinase, near the catalytic site and reacts with free and receptor bound, single and two chain urokinase and the B-chain fragment. Monoclonal mouse anti-PAI-1 IgG (no. 3785; American Diagnostic) had been raised against purified active PAI-1 secreted by the human melanoma cell line. A polyclonal sheep anti- PAI-2 IgG (Biotech Australia, NSW, Australia) was used to detect both the high molecular weight (60 kDa) form of PAI-2 and the low molecular weight (48 kDa) form of PAI-2. Monoclonal mouse antibodies against the proliferation cell nuclear antigen (PCNA) (Zymed Lab. BioScientific, Australia) and Ki-67 (Zymed Lab. BioScientific, Australia) which reacts with Ki-67, a cell proliferation marker for all active stages of the cell cycle (Gl, S, M, and G2), but not the GO stage were also used.

Immunohistochemical methods All stages of the immunostaining procedures were carried out at room temperature. Tissue sections were dewaxed in xylene 3 times at 5 minutes for each. Prior to immunoperoxidase staimng, endogenous peroxidase activity was quenched by incubating the tissue sections with 3 % H₂O₂ for 20 minutes. All the non-specific bindings were blocked by 0.1 % bovine serum albumin (BSA) with 10% swine serum. Antibodies to tPA, uPA, PAI-1 and PAI-2 were used at a dilution of 100 μg/ml, 3 μg/ml, 5 μg/ml and 10 μg/ml in PBS containing 0.1 % BSA and allowed to incubate for 60 minutes. For antibodies against PCNA (diluted in 1:100) and Ki- 67 (diluted in 1:2), tissue sections were treated with target retrieval solution (DAKO, USA) by heating at 95°C for 30 min. After incubation with the primary antibodies, sections were rinsed with PBS solutions. Sections were then incubated with a biotinylated swine-anti- mouse, rabbit, goat antibody (DAKO Multilink, CA, USA) for 15 minutes, and then incubated with horseradish periodase-conjugated streptavidin for 15 minutes. Antibody complexes were visualised after the addition of a buffered diaminobenzidine (DAB) substrate for 4 minutes). The reaction was stopped by immersion and rinsing of sections in PBS. Sections were then lightly counterstained with Mayer's haematoxylin and Scott's blue for 40 seconds each, in between 3 minute rinses with running water. Following this, they were dehydrated with ascending ethanol, cleared with xylene and mounted with coverslip using DePeX mounting medium (BDH Laboratory Supplies, England).

Controls for the performance of the immunostaining procedures included conditions where the primary antibody or the secondary (anti-mouse IgG) antibody were omitted and an irrelevant antibody was used as a control. To ensure that the procedure itself was not causing non-specific staining, various safeguards were used. These included elimination of the primary incubation step, in the presence of all other steps; and normal primary antibody incubation followed by elimination of either the secondary antibody or one of the other subsequent detection steps. Sections were viewed and photographically recorded using an Olympus System Microscope (Model BX50, Tokyo, Japan).

Collection of periodontal wound healing fluid

The periodontal wound healing fluid was collected from the gingival crevice of the wound sites at different healing periods before the animal was killed. The collection sites were cleaned by removing the obvious supragingival plaque with a curette and the area was carefully isolated from saliva with cotton rolls, gently air-dried. Sterile 2 x 10 mm strips of Whatman No. 1 filter paper (Whatman International Ltd, Springfield Mill, Maidstone,

England) were inserted gently into the gingival crevice of the wounded areas for 0.5 min.

Care was taken in order to avoid mechanical injury of the tissues. The portion of the strip containing the fluid sample was cut off and placed individually into a microcentrifuge tube containing 100 μl of Tris buffer (12 mM Tris, 0.1 M NaCl, 0.05% Tween 20). The samples were vortexed and stored at room temperature for 1 hour. The filter paper strip was discarded and the sample solution was frozen at -20°C prior to analysis.

Enzyme Immunoassay

Before analysis, the wound healing fluid samples were thawed at room temperature for 30 min. and vortexed for 10 min. Each sample was then analysed both for t-PA and PAI-2. t-PA antigen levels were measured using the ELISA kit (IMUBIND total t-PA stripwell

ELISA, American Diagnostica Inc. Greenwich) which is intended for quantitative determination of human tissue type plasminogen activator antigen and the immunoreactivities of single-chain and two-chain-tPA in complex with, PAI-1, and PAI-2. The standard curve was linear between 0 and 30 ng/ml. 20 μl of each sample was used for the assay and each sample was processed in duplicate. The absorbency value for each sample was used for the calibration of t-PA concentration. The t-PA ELISA method was performed briefly as below.

After reconstitution of wells, 20 μl of each test sample was added to the wells and incubated for 1 hour. 50 μl of the conjugate (HRP labelled anti-tPA) was added to each well and incubated for 15 minutes. After 5 times wash, color was produced by 1,2 phenylenediamine dilhydrochloride (OPD) and the absorbance was measured at 490 nm. Levels of PAI-2 antigen were also measured on 20 μl samples using an ELISA assay (Biotech, Sydney, Australia) and the results were calculated on duplicate samples. This assay uses rabbit polyclonal antibodies, and detects glycosylated and non-glycosylated PAI- 2, as well as PAI-2 complexed with uPA or tPA. A standard curve was determined using yeast recombinant human PAI-2 and was linear over the range of 0 to 40 ng/ml. For PAI-2 ELISA assay, 20 μl of sample was added to the reconstitution well and incubated for 2 hours. After 10 times wash, 100 μl of the conjugate (HRP labelled anti-PAI-2) was added to the well and incubated for 2 hours. The colour was produced by peroxidate substrate 3,3' ,5,5'-tetramethylbenzidine (TMB) and the absorbance was measured at 650 nm.

Results were expressed as ng/ml per site in 0.5 min collection time sample that washed out in 100 μl of Tris buffer. Control wells in each plate were included which contained no sample or standard antigen in order to calculate background binding.

Testing of bioadhesive gel formulations for delivery of active to periodontal pocket A preferred administration form for the active of the invention is a bioadhesive gel formulation. Accordingly, various such gel formulations were tested for their compatibility with an active according to the invention; ie tested for their (a) adhesive qualities, ie retention in the periodontal pocket, (b) ability to maintain the activity of PAIs at certain temperatures, and (c) ability to allow the release of PAIs to the periodontal tissue cells. The testing was conducted according to practices standard in the art using PAI-2 as representative of an active of the invention. It will be appreciated that similar procedures may be used in order to assess the suitability of a particular formulation for the delivery of different PAIs, without having to utilise any inventive activity.

Briefly, study gels were prepared by mixing in PBS at room temperature a designated bioadhesive agent, preservative, and sterile PAI-2 to a final concentration of lmg PAI-2 per ml of gel; bioadhesives and preservatives used are seen from Figure 6. The preservative parabens contained 0.18% methyl parabens and 0.02% propyl parabens. Where chlorhexidine/EDTA was used as a preservative, the agent contained 0.025% chlorhexidine and 2.5mM EDTA.

For stability studies, aliquots of 1ml of gel were stored at designated temperatures (room temperature for all studied gels; for pluronic F127 a study of storage at 4 C was also conducted, as seen in figure 8). At specified times (see Figure 7 and Figure 8), one or more aliquots were removed and the PAI-2 activity measured using the Spectrozyme assay. This assay, which will be readily recognised by those of skill in the art to which this invention relates, uses Spectrozyme^® (of American Diagnostica Inc) containing an amino acid recognition sequence for the protease uPA, linked to a chromogenic compound. uPA introduced to the assay chamber cleaves Spectrozyme liberating the chromogen (para- nitroanilide (pNA)), which may be detected in a spectrophotometer at a wavelength of 405 nm. The acitivity of PAI-2 is detected by inhibition of the action of uPA. The Spectrozyme assay was conducted according to the manufacturers recommendations.

In vitro release studies were conducted wherein a gel containing PAI-2 was placed in the top chamber of a two compartment vessel. The bottom chamber contained PBS, and the two chambers were separated by a microporous membrane. Samples were removed from the lower chamber and assayed for PAI-2 activity using the Spectrozyme assay. Detection of PAI-2 activity in the lower chamber indicated release of PAI-2 from the upper chamber (Figures 9 and 10).

Retention of gel formulations in the periodontal pocket is detected in humans, and other subjects, by observation, after a gel is inoculated into the periodontal pocket of a patient from a syringe with a smooth, bent tip to allow delivery. Typically, the gel is observed for a period of one hour post inoculation into the periodontal pocket.

Bioburden (as referred to in the table of Figure 6) was measured as the ability of a preparation to reduce an inoculum of at least lOVml of standard bacteria species, and Aspergillus niger, by 2 logs in 14 days of incubation. Results

Histomorphology of periodontal wound healing

Periodontal tissue biopsies including gingival, alveolar bone and tooth were taken at different time intervals after wounding. Haematoxylin and eosin staining of these sections revealed early inflammatory cells infiltration, granulation tissue formation and tissue remodelling. At day 1 and 3 (figure la), most inflammatory cells infiltrated the gingival tissue and sulcus. By day 1 a fibrin clot had formed around the root side of the surgical wound areas. Polymorphonuclear neutrophils (PMNs) were observed in and around the fibrin clots. At day 3. granulation tissue had begun to form close to the gingival sulcus. The cells' shapes in granulation tissue were fibroblast-like and the cell density was high.

From day 3 to day 14 cell proliferation was noted in this area by immunohistochemical staimng of nuclear proliferation antigen, (PCNA and Ki 67 (figure lb)).

Monocytes/macrophages dominated in the wound areas during this stage. A new attachment apparatus began to form between days 7 to 14, which included both connective tissue and epithelial attachment to the root surface. All these parameters are consistent with a regular time course of periodontal wound healing.

The expression of t-PA and u-PA in periodontal healing wounds

In all the sections, t-PA staining was found only in the fibrin clot at the early stages of wound healing. Faint staining for t-PA was noted in the day 3 granulation tissue cells, but no staining was found in the inflammatory cells that infiltrated into gingival sulcus and gingival tissue (figure 2d). u-PA was diffusely distributed in the whole period of wound healing. Very strong expression was noted in the early infiltrated cells from day 1 (figure lc) to day 7. The expression of u-PA in granulation tissue cells was strong and the staining density increased from day 3 to day 7, then decreased from day 7 to day 28. During the time of new attachment formation, u-PA staining was noted in the attached cells onto the root surface (figure 2b). During the stage of fibrin resolution (day 3 to day 7), the inflammatory cells and fibroblasts near the fibrin clot expressed u-PA very strongly (figure

2a). By day 3, u-PA was also strongly localised in the basal epithelial cells at the edge of tissue wounding. A very strong expression of u-PA was also found in endothelial cell in the newly formed blood vessels in the granulation tissue (figure 3a). PAI-1 and PAI-2 in periodontal healing wounds

During the early stages of periodontal wound healing, PAI-1 and PAI-2 strongly localised in the provisional matrix (figure Id). The expression of PAI-1 and PAI-2 in inflamed cells was very intense during the early stages from day 1 and day 3 (figure lb). The staining decreased in these cells from day 5 and day 7, but some staining in infiltrated cells could be found. In the granulation tissue cells, the expression of PAI-1 was stained stronger in day 3 and day 5, then weakly expressed in day 7 and after. PAI-2 was much stronger stained in day 5 and day 7 tissue slices than the day 3 and day 14 slices in these granulation tissue cells. In the fibroblasts that had recently attached to the root surface, a strong expression of PAI-1 and PAI-2 was noted. This special expression was also found in the epithelial attachment to the root surface (figure 2c).

t-PA and PAI-2 levels in wound healing fluid Wound healing fluid was collected by filter paper and analysed by ELISA. The results showed that t-PA in wound fluid increased from day 1 to day 7, then the level decreased until day 28 (figure 4). PAI-2 was present at a low levels during the whole wound-healing period with the same pattern as t-PA, which was present in high levels during the early stages of wound from day 1 to day 7, and then decreased to a low level during the tissue remodelling stage from day 7 to day 28 (figure 5).

The results described herein elucidate for the first time that the expression of plasminogen activators by different cell types in periodontal wound healing has concerted roles in the formation of fibrin matrix and tissue remodeling. The results demonstrate that during periodontal wound healing the strong expression of PAI-1 and PAI-2 in provisional matrix leads to the formation of the fibrin clot. The fibrin clot provides an early, primitive form of extracellular matrix, which potentiates the migration of inflammatory cells into the periodontal sulcus and the lesion. The high amounts of u-PA secreted by granulocytes and macrophages that infiltrate into periodontal wound area, together with the t-PA derived from blood vessel disruption and plasma extravasation, lead to the ultimate dissolution of the fibrin clot. The localisation of u-PA, PAI-1 and PAI-2 in new attached fibroblasts and epithelial cells on the root surface indicated that the plasminogen activators play a significant role in reepithelialisation and the formation of new periodontal attachment.

Selection of bioadhesive gel formulation for delivery of PAI-2 to the periodontal pocket It will be appreciated that optimal bioadhesive pharmaceutical compositions are selected for use in the treatment of periodontitis by PAIs on the basis of, for example, (a) adhesive qualities, ie retention in the periodontal pocket; (b) maintaining the activity of PAIs at certain temperatures; and (c) allowing the release of PAIs to the periodontal tissue cells. Accordingly, the compatibility of various bioadhesive formulations with an active representative of the invention, PAI-2, were studied. Figures 6 to 10 show, by way of example, the results of these studies.

The results obtained indicate that certain bioadhesives, such as polycarbophil (Figures 6 and 7), may not be compatible with PAI-2, while other bioadhesives may be so. It will be appreciated that a finding herein that certain bioadhesive formulations are not optimally compatible with PAI-2 does not necessarily mean that those formulations will not be compatible with an alternative PAI active, and vice versa. A person of ordinary skill in the art would readily be able to ascertain which formulations were compatible with which actives using the procedures described herein.

The results illustrated by Figure 8 indicate that the storage temperature of a gel formulation according to the invention may affect the stability of the active therein. The present studies indicate that where PAI-2 is formulated with Pluronic F127 storage at room temperature is optimal. Again, the optimal storage temperature of a particular composition may vary depending on the active therein and the bioadhesive gel formulation used. However, those of ordinary skill in the art will appreciate such variation and will be able to conduct routine studies, such as those described herein, to elucidate an optimal storage temperature.

Results obtained from studying the formulations ability to allow sustained release of the active, PAI-2 in this example, are shown in Figure 9 and 10. Slow, steady release as shown by carbopol, xanthum, poligrip or 30% pluronic F127, are preferable deliveries. However, it will be readily appreciated by those of ordinary skill in the art that preferred deliveries may vary depending on the precise gel formulation/active combination.

Composition Examples Preferred compositions encompassed by the present invention are made according to the examples provided below. Further specific examples will be apparent to the reader from the experimental protocols described herein before and with reference to Figure 6.

In one example, a gel formulation is prepared consisting of 1.8% (w/v) hydroxyethyl cellulose, 10% (v/v) propylene glycol and 0.02% (v/v) polysorbate 80. These components are mixed in PBS and filter sterilised under pressure. To this gel, 1 part per 100 of sterile PAI-2 solution of 5mg PAI-2 per ml of PBS is added with mixing at room temperature to provide a final concentration of active (PAI-2) of 50 g/ml.

In another example, the above gel is cooled to 4 C in a vessel surrounded by ice and the bioadhesive Pluronic F127, also cooled to 4 C, is added to a final concentration of 17% (v/v) and mixed to consistency. Similarly cooled PAI-2 solution is added as described above. After preparation at 4 C the composition is preferably stored at room temperature.

In preparing gel preparations as exemplified above, mixing is generally complete after 15 minutes at room temperature, or 45 minutes at 4 C.

Treatment Example

Subjects having a periodontal condition can be administered an active, according to the invention, in a suitable composition, to show an improvement in periodontal tissue regeneration and attachment, as indicated in the following non-limiting examples.

A gel having a pharmaceutical composition as described in the preceding "Composition

Examples" section, including for example, PAI-2 at a concentration of 50μg/ml, is administered to a patient from a syringe. The syringe is attached to a 21 gauge irrigation needle with a smooth blunt end, bent to a 90 angle 5mm from its tip. PAI-2 gel is delivered directly to the periodontal pocket using this syringe and needle attachment.

0.1ml of gel (PAI-2 50μg/ml of gel) is administered to each periodontal pocket, once per week, for 12 weeks. Over this period, an improvement in periodontal tissue regeneration can be observed and in a number of cases complete periodontal tissue regeneration and attachment will occur.

Similar results can be observed using compositions having alternative concentrations of active (for example PAI-2), by administering alternative amounts of such a composition, and varying the duration of treatment. Those skilled in the art will readily be able to derive alternative treatment regimes based on standard procedures known to such persons and the information provided in this specification.

In summary, the results described herein are the first to indicate that in periodontal wound healing, the plasminogen activator system is involved in the formation of fibrin matrix and its replacement by granulation tissue, and is also involved in the attachment of soft tissue to the root surface during the later stages of wound repair, ie periodontal tissue attachment.

Concomitant with these findings, the inventors have identified that inhibitors of the plasminogen activator system, and in particular PAI-1 and PAI-2, are of benefit in the artificial regulation of periodontal tissue formation, including periodontal attachment, and more particularly, the therapeutic and/or prophylactic treatment of a periodontal condition in a mammal; for example, gingivitis, periodontitis or gum injury. Further, the inventors have illustrated various bioadhesive gel formulations suitable for use in the methods of the invention.

The invention has been described herein, with reference to certain preferred embodiments, in order to enable the reader to practice the invention without undue experimentation. However, a person having ordinary skill in the art will readily recognise that many of the components and parameters may be varied or modified to a certain extent without departing from the scope of the invention. It will be understood that the invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features. Furthermore, titles, headings, or the like are provided to enhance the reader's comprehension of this document, and should not be read as limiting the scope of the present invention.

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

Finally, the reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in the field of endeavor.

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Claims

CLAIMS:

1. A method of regulating periodontal tissue formation in a mammal comprising administering to said mammal an effective amount of one or more PAIs or functional derivatives, equivalents, analogues, homologues or mimetics thereof for a time and under conditions sufficient to modulate the functional activity of any one or more components of the periodontal tissue formation pathway.

2. A method as claimed in claim 1 wherein the method induces, enhances or otherwise up- regulates periodontal tissue formation in a mammal.

3. A method as claimed in either of claims 1 or 2 wherein the method results in periodontal attachment.

4. A method as claimed in any one of claims 1 to 3 wherein the components are one or more molecules or cells which function during periodontal tissue formation.

5. A method as claimed in claim 4 wherein the component is an enzyme.

6. A method as claimed in claim 5 wherein the components are plasminogen activator and/or plasmin and the modulation is inhibition.

7. A method as claimed in any of claims 1 to 6 wherein the one or more PAIs are PAI-1 and/or PAI-2.

8. A method of treating a mammal comprising administering to said mammal an effective amount of one or more PAIs or functional derivatives, equivalents, analogues, homologues or mimetics thereof for a time and under conditions sufficient to modulate any one or more components of the periodontal tissue formation pathway.

. A method as claimed in claim 8 for the therapeutic and/or prophylactic treatment of a condition in a mammal, which condition is characterised by damage, breakdown or other form of degradation of periodontal tissue and wherein said modulation results in the induction, enhancement or up-regulation of periodontal tissue formation.

10. A method as claimed in claims 9 wherein said modulation results in periodontal attachment.

11. A method as claimed in claims 9 or 10 wherein the condition in a mammal is at least one of the disease conditions gingivitis, periodontitis or gum injury.

12. A method as claimed in claims 9 or 10 wherein said gum injury is caused by major or minor surgery, infection, or pregnancy-related tissue deterioration.

13. A method as claimed in any of claims 8 to 12 wherein the components are one or more molecules or cells which function during periodontal tissue formation.

14. A method as claimed in claim 13 wherein the component is an enzyme.

15. A method as claimed in claim 14 wherein said components are plasminogen activator and/or plasmin and said modulation is inhibition.

16. A method as claimed in any one of claims 8 to 15 wherein the one or more PAIs are PAI-1 and/or PAI-2.

17. Use of an agent adapted to modulate the functional activity of any one or more components of the periodontal tissue formation pathway wherein said agent is a PAI or a functional derivative, equivalent, homologue, analogue or mimetic thereof.

18. Use as claimed in claim 17 wherein said modulation results in inducement, enhancement or up-regulation of periodontal tissue formation.

19. Use as claimed in claim 18 wherein said modulation results in periodontal attachment.

20. Use of one or more PAIs or functional derivatives, homologues, analogues, chemical equivalents or mimetics thereof in the manufacture of a medicament for modulating periodontal tissue formation.

21. Use as claimed in claim 20 wherein the one or more PAIs are PAI-1 and/or PAI-2.

22. A pharmaceutical composition adapted to modulate the functional activity of any one or more components of the periodontal tissue formation pathway comprising one or more PAIs or functional derivatives, homologues, analogues, chemical equivalents or mimetics thereof, together with one or more pharmaceutically acceptable carriers and/or diluents.

23. A pharmaceutical composition as claimed in claim 22 wherein the one or more PAIs are PAI-1 and/or PAI-2.

24. A pharmaceutical composition as claimed in either of claims 22 or 23 wherein the composition is adapted for topical or injectable administration.

25. A pharmaceutical composition as claimed in claim 24 wherein where the composition is adapted for topical administration it further comprises: at least one compatible bioadhesive compound.

26. A pharmaceutical composition as claimed in claim 25 wherein said at least one compatible bioadhesive compound is chosen from the group consisting carboxymethyl or hydroxyethyl cellulose, xanthum gum, chitosan, carbopol 974P, poligrip, or pluronic F127.